The survival of every species depends on the faithful inheritance of genetic information. Essential to this process are the accurate movement and positioning of chromosomes and plasmids to daughter cells at cell division. The ultimate goal of this proposal is to elucidate at the atomic level, basic mechanisms of DNA segregation. Plasmid partition (par) systems represent excellent model systems to study the molecular mechanisms of DNA partition because they require only three components: a centromere site, and proteins, a polymer-forming protein and centromere-binding protein. The best understood par systems contain polymer-forming proteins called ParM and centromere-binding proteins called ParR. This proposal focuses on one such par system from Staphylococcus aureus multi-drug resistance plasmid pSK41. In the first step of partition, ParR binds cooperatively to the centromere to form a large, wrapped nucleoprotein complex called the segrosome. ParM-ATP is then recruited to paired segrosomes, stimulating ParM filament formation. The growing ParM filament ultimately pushes the paired plasmids apart. Vital questions that remain to be resolved are what type of large nucleoprotein complex is formed by ParR binding to its centromere, how this serves to recruit ParM and how ParM is stimulated to form filaments by ATP and the segrosome? In addition, the pSK41 system is unique among par systems in that, in addition to ParR, it requires a novel, chromosomally encoded protein, ArtA, for full par transcription autoregulation. Our recent structure determination of pSK41 ParR bound to its centromere reveal the first structure of a segrosome and make important predictions about how segrosomes recruit and stabilize filament formation. In this grant proposal we will build on this recent progress towards a full elucidation of pSK41 partition and its regulation with the following Specific Aims: (1) Fully elucidate the mechanism of transcription regulation of the pSK41 par operon by determining the structure of the ArtA-DNA complex (2) Clarify the mechanism of plasmid separation via structural and biochemical studies on the key conformational states of pSK41 ParM (apoParM, ParM-ADP and the ParM-ATP) as well as a structure of ParR-centromere-ParM-AMP-PCP. We will also elucidate the structure of the ParM filament and examine its interaction with the segrosome using electron microscopy, biochemistry and cellular studies. Importantly, pSK41, harbored in S. aureus, confers resistance to multiple antibiotics, including the drug of last resort, vancomycin. Such multi-drug resistant S. aureus strains are becoming a serious threat to human health. Indeed, recent reports indicate that multidrug resistant S. aureus now kills more individuals in the U.S. than HIV/AIDS. Thus, the work described in this proposal will provide potential points of therapeutic intervention against such multi-drug resistant S. aureus strains by targeting the essential par proteins and complexes required for maintenance of multi-drug resistance determinants. PUBLIC HEALTH RELEVANCE: Multidrug resistant Staphylococcus aureus now kills more individuals in the U.S. than HIV/AIDS. The pSK41 plasmid, which is harbored in S. aureus, carries many of these multi-drug resistant genes. Retention of this plasmid demands it be accurately segregated during cell division;a process dependent on its par system. Thus, understanding the structural basis for DNA segregation by this par system will provide several points of potential therapeutic intervention against pSK41 harboring multidrug resistant S. aureus strains.